We have obtained evidence that the PC system plays a crucial role in the regulation of the pulmonary inflammation accompanying tuberculosis. In particular, mice with a mutation in the gene encoding TM (TMpro/pro mice), making them less capable of generating APC in their lungs, were found to mount uncontrolled lung inflammation during tuberculosis [4]. This study left unanswered which components of the PC system are important for this phenotype and whether administration of the active end product of this pathway, APC, can positively influence the pulmonary response to tuberculosis. Therefore the objectives of this project are:(1) To determine the role of the endogenous PC system in the host response to tuberculosis1.1: Expression of the PC system: To which extent is the expression of the PC system altered in the lungs of patients with tuberculosis? To which extent is the expression of the PC system altered in mice with experimentally induced tuberculosis?1.2: Function of the PC system: What are the roles of specific components of the PC system in tuberculosis mice?(2) To determine the effect of inhaled recombinant APC in tuberculosis.2.1: Effect of recombinant APC: What are the effects of inhaled recombinant APC on the host response to tuberculosis? 2.2: APC mechanism of action: What is the relative contribution of the anticoagulant and cytoprotective properties to the effect of APC on the host response to tuberculosis?
Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a major global health problem. The host response to tuberculosis is characterized by an orchestrated inflammatory reaction in the lungs involving multiple cell types and cytokines and resulting in the formation of granulomas. The Protein C (PC) pathway has been implicated as an important regulator of coagulation and inflammation. The PC pathway is initiated by the binding of thrombin to thrombomodulin (TM) after which the TM-thrombin complex activates PC to Activated Protein C (APC). Thrombin/TM dependent activation of PC is strongly augmented by the endothelial PC receptor (EPCR). The biological effects of APC (the end product of the PC pathway) are pleiotropic, and can be divided in anticoagulant and cytoprotective effects. Dissociation of APC from EPCR allows expression of APC s anticoagulant activity (by inactivation of clotting factors Va and VIIIa), whereas retention of APC bound to EPCR allows APC to express multiple direct cellular cytoprotective activities (including alteration in gene expression, anti-inflammatory and anti-apoptotic effects, and protection of endothelial and epithelial barrier functions); these latter effects are dependent on EPCR and mediated by protease activated receptor 1 (PAR1). Most research on the PC pathway has focused on its role in the circulation, especially after severe injury and/or infection. Recent evidence, in part derived from studies performed in the host laboratory, point to an important role for the PC pathway in the lung. In this regard it is of importance that all essential elements of the PC pathway are constitutively expressed by resident cells in the pulmonary compartment. The current grant proposal is based on our recent discovery that mice with a point mutation in their TM gene (TMpro/pro mice), making them substantially less capable of generating APC in their lungs, display uncontrolled lung inflammation during tuberculosis [Weijer et al. BLOOD 2005]. This unexpected finding left several questions unanswered that will be addressed in this project. The objectives of this project are (1) to determine the role of the endogenous PC system in the host response to tuberculosis and (2) to determine the effect of inhaled recombinant APC and its mechanisms of action in tuberculosis.We will study the influence of tuberculosis on the expression of the PC system and the accompanying procoagulant and inflammatory response in bronchoalveolar lavage fluid obtained from patients who undergo bronchoalveolar lavage for suspected tuberculosis (but with negative sputum smears). This prospective study will be done in the Sapprasithiprasong hospital, Ubon Ratchathani (east Thailand), in close collaboration with the Welcome Trust unit in Bangkok. The host laboratory has an existing collaboration with the Welcome Trust and successfully conducted clinical studies in Ubon on melioidosis in the recent past. We chose to perform this study in Thailand, since we expect that such an investigation is unlikely to be successful in the Netherlands due to the limited number of feasible patients. In addition, we will examine TM, EPCR and PAR1 expression in (stored) human lung biopsies of 10 patients with culture proven tuberculosis and (as a control) 10 noninfected patients (normal tissue surrounding tumor) using immunohistochemistry. The functional role of the endogenous PC pathway in tuberculosis will be studied using our established model for pulmonary tuberculosis in mice treated with an anti-PC antibody and in genetically modified mice with an impaired or absent function of TM, EPCR or PAR1. In addition, the effect of inhaled recombinant APC will be determined in this model; furthermore, to obtain insight into the mechanism of action of APC during tuberculosis, we will also administer APC mutants that lack anticoagulant activity to wild-type mice and wild-type APC to mice deficient for EPCR or PAR1. The proposed studies will provide in-depth insight into the role of the PC pathway and the effect of APC in tuberculosis. |
